Five ETS family members, ELF-1, ETV-4, ETV-3L, ETS-1, and ETS-2 upregulate human leukocyte-associated immunoglobulin-like receptor-1 gene basic promoter activity

Determination of the minimal promoter region of LAIR-1

The minimum sequence required for promoter activity was determined by generating a series of luciferase reporter plasmids containing truncated DNA fragments of the LAIR-1 promoter sequence and transiently transfecting into HEK293T cells. The promoter activity of these truncated DNA fragments was measured by luciferase assay (Fig. 1). The luciferase activity of pGL-lair1-p2 (-1684/-8) had relatively higher promoter activity and showed a 6.65-fold increase compared with the empty pGL4.17 vector, indicating a functional promoter in the -1684 to -8 region relative to TSS of LAIR-1 gene. When the DNA sequence was deleted to position -875, the promoter activity with pGL-lair1-p3 (-875/-8) decreased significantly compared with the pGL-lair1-p2 (-1684/-8) (Fig. 1), this result demonstrates that positive regulatory elements may be located in the -1684 to -875 region. 5’ deletions from position −875 to -256 did not significantly change activity; A further deletion to -162 significantly decreased the promoter activity of pGL-lair1-p7 (-162/-8) when compared with pGL-lair1-p6 (-256/-8), indicating the presence of one positive regulatory element may be located in this deleted region. Analysis of luciferase activities of pGL-lair1-p7 (-162/-8), pGL-lair1-p8 (-93/-8), and pGL-lair1-p9 (-43/-8), demonstrates the possible presence of one positive regulatory element and one negative regulatory element at the -93 to -43, and -162 to -93 regions, respectively. These results indicate that pGL-lair1-p6 (-256/-8) has the basic promoter activity, that the minimal promoter of the LAIR-1 gene may be located at the -256 to -8 region relative to TSS, and that this region possibly contains one negative and two positive regulatory elements.

Regulation of LAIR-1 basic core promoter by transcription factors ETS family

The putative transcription factor binding sites within the basic promoter of lair-1 sequence were identified using online PROMO software (http://alggen.lsi.upc.es/cgi-bin/ promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and JASPAR database (http://jaspar.genereg.net. net/). After we ranked them in descending order according to the relative scores, about 50% of the top 30 predicted transcription factors belonged to the ETS family, with the ETS-like, ELK-like and ELF-1-like subfamilies were dominating. Therefore, we chose several transcription factors from these three ETS subfamily (ETS-like subfamily: ETS-1, ETS-2, ERG and ETV3L; ELK-like subfamily: ELK-4 and ETV-4; ELF-1-like subfamily: ELF-1 and ELF-2.) to determine their potential effects on the regulation of the lair-1 promoter activity. We also chose two EHF-like family members (ELF-3 and ELF-5), which has been reported to be involved in ovarian cancer, to detect whether they can regulate LAIR promoter activity [24]. We first investigated whether ETS-1 and ETS-2 could regulate the minimal promoter activity of LAIR-1. We co-transfected with ETS-1 or ETS-2 expression vectors, and pGL-lair1-p6 (-256/-8), pGL-lair1-p8 (-93/-8), pGL-lair1-p9 (-43/-8) or pGL-lair1-p10 (-162/-92) into HEK293T cells, we found ETS-1 and ETS-2 increased pGL-lair1-p6 (-256/-8) activity to 7.67- and 37.95- fold, respectively. ETS-2, but not ETS-1, also increased the activity of pGL-lair1-p8 (-93/-8) and pGL-lair1-p10 (-162/-92) (Fig. 2). The results confirmed that -256/-8 had the basic promoter activity which can be regulated by ETS-1 and ETS-2. We further examined the effects of other ETS members including ELF-1, ELF-2, ELF-3, ELF-5, ELK-4, ERG, ETV-3L, and ETV-4, on the LAIR-1 basic promoter activity. As shown in Figure 3, ELF-1, ETV-4, ETV-3L, ETS-1 and ETS-2 significantly enhanced the basic promoter activity, and ETS-2 was the most effective transcription factor.

Effect of ELF-1, ETV-4, ETV-3L, ETS-1 and ETS-2 on LAIR-1 gene expression in HO8910 cells

According the results of the luciferase assays, we know that ELF-1, ETV-4, ETV-3L, ETS-1 and ETS-2 can enhance LAIR-1 basic promoter activity. We further studied the effect of these transcription factors on the regulation of LAIR-1 gene expression in ovarian cancer cell HO8910, which we have used in our previous study to investigate the function of LAIR-1 [15]. As shown in Figure 4, five ETS members had distinctive regulation efficiency on LAIR-1 expression in HO8910. All five factors can enhance LAIR-1 protein expression, while ETS-1, ETS-2 and ETV-4 can significantly increase LAIR-1 mRNA expression; Consistent with the results of the luciferase assays, ETS-2 is the most efficient transcription factor, which can significantly increase the LAIR-1 mRNA expression in HO8910.

Binding of ETS-2 to the LAIR-1 basic promoter

Because ETS-2 can upregulate both LAIR-1 basic promoter activity and gene expression with high efficiency, suggesting there may be a direct interaction between ETS-2 and LAIR-1 basic promoter. Based on the analysis results obtained using online software PROMO and JASPAR database, three highly potential ETS-2 binding sites within the LAIR-1 basic promoter, A-site (5’-GGGAAT-3’, at position -176 to -171), B-site (5’-CTTCCT-3’, at position -145 to -140) and C-site (5’-ACATCCTGT-3’, at position -84 to -76), were identified (Fig 5A). In order to investigate the importance of these three sites in LAIR-1 basic promoter activity, we constructed a series of DNA report plasmids with deletion mutations of these sites and co-transfected HEK293T cells with these plasmids and ETS-2 expression vector. As shown in Figure 5B, the LAIR-1 promoter activity of pGL-lair1-p6, which has all sites intact, was increased by nearly 60-fold when compared with the basic pGL4.17. Disruption of either site significantly reduced the activity. These results indicate that all three ETS-2 binding sites are necessary for maximal promoter activity; however, the A-site is least effective, while the B-site and C-site are the most effective.

We used CHIP assays to examine in vivo the association of ETS-2 with the LAIR-1 gene basic promoter region. Jurkat and COC1 cells were used because of their high expression of LAIR-1. As shown in Figure 6A, the PCR product obtained with CHIP DNA using the -256 to -8 region sequence primers indicated a specific interaction between ETS-2 and a chromatin fragment containing this promoter region. This interaction was observed both in Jurkat cells and COC1 cells. We further used EMSA assay to investigate whether ETS-2 could physically bind in vitro with the above three binding sites. As shown in Figure 6B-D, the nuclear protein from Hela cell expressing high level of ETS-2 bound to B-site and C-site (Fig. 6C, lane 2; Fig. 6D, lane 2), but not to the A-site (Fig. 5A, lane 2). Competition assays using an excess of unlabeled probe verified the specificity of both B-site and C-site probe/DNA interaction. EMSA results further confirmed both B and C-site are effective binding sites for ETS-2.

Plasmid construction of truncated and deletion mutants of the LAIR-1 basic promoter

Based on our previous study bioinformatics analysis [16], we used a DNA sequence of 2485 bp(-2493 to -8)upstream from the translational start site (TSS) in the LAIR-1 gene as the candidate for identifying the promoter region. The TSS was defined as the +1 position. This 2485 bp fragment was amplified by PCR using genomic DNA extracted from human peripheral blood mononuclear cells and cloned into a pGL4.17-Luc luciferase reporter vector (Promega) using the restriction endonuclease KpnI and XhoI sites. The primer sequences were 5’-CGG GGTACC CCC CTG TCT GTC CAC GAG ATC CAA-3’ (underlined regions denote the KpnI recognition sequence sites) and 5’-TCC GCTCGAG GT CCC AGC AGT GCA GCC TGG-3’ (underlined regions denote XhoI recognition sequence sites). This report plasmid was designated as pGL-lair1-P1. A series of truncated fragments of the regulation region of the LAIR-1 gene were created by PCR amplification using the primers listed in Table 1 and pGL-lair1-P1 as template, and these were inserted into a pGL4.17-Luc luciferase reporter vector using the same restriction endonuclease Kpn I and Xho I sites. These plasmids containing the truncated fragments were designated as pGL-lair1-P2 to pGL-lair1-P15. Plasmids harboring deletion mutations within the core LAIR-1 promoter were constructed with a site-specific mutagenesis protocol using an overlap extension PCR and the primers listed in Table 2 [32]. DNA sequences of all the clones (the wild-type and mutated promoters) were verified by DNA sequencing. Expression vectors of transcription factor, ETS-1 (pcDNA-ETS1), ETS-2 (pSPORT-ETS2) ELF-1 (pCDB-ELF1), ELF-2 (pCDB-ELF2), ELF-3 (pCDB-ELF3), ELF-5 (pCDB-ELF5), ELK-4 (pCDB-ELK4), ERG (pCDB-ERG), ETV-3L (pCDB-ETV3L), ETV-4 (pCDB-ETV4) were purchased from Sino-GenoMax Company Limited (Beijing, China).

Transient cell transfection

Promoter activity was analyzed using a promoterless pGL4.17-basic vector as a negative control, and plasmid pRL-TK (Promega) was cotransfected as an internal control. All plasmids were propagated in Escherichia coli DH5α and isolated using a QIAprep spin miniprep kit (Qiagen). HEK293T cells were plated in 96-well plates 24 h before transfection, and triple wells were set for each group. A 100 ng of pGL4.17 vector containing different truncated DNA fragments and 4.0 ng amount of pRL-TK vector were cotransfected for each well using Vigofect (Vigofect Inc. Beijing, China), according to the manufacturer’s instructions. The regulation of the LAIR-1 promoter by ETS family members was investigated by cotransfecting an expression vector of transcript factors using Vigofect; the corresponding basic vector was used as a negative control.

Luciferase assay

After 48 h of transfection, cell lysates were prepared for the measurement of luciferase activity using the Dual-Luciferase^® Reporter Assay System (Promega). The promoter activity was calculated from the chemical luminescence intensity ratio of firefly: Renilla luciferase for each construct; and then compared with the activity of vector pGL4.17 luciferase. The data were presented as the mean ± S.D from three independent experiments.

Protein immunoblotting and RNA quantification

Effects of ELF-1, ETV-4, ETV-3L, ETS-1, and ETS-2 on LAIR-1 gene expression were analyzed using Quantitative real time PCR (qRT-PCR) and Western blotting analysis. An ovarian cancer cell line HO8910 was used in the assay. Cells were plated in 6-well plates 18 h before transfection, and triple wells were set for each group. 1μg transcription factor expression vector was transfected for each well using Vigofect (Vigofect Inc. Beijing, China), according to the manufacturer’s instructions. Cells were collected after 48 h transfection. Total RNA was extracted using Trizol reagent (Invitrogen, USA) and reverse-transcribed to cDNA using the Revert Aid First Strand cDNA Synthesis Kit (Fermentas, Canada). qRT-PCR was performed using Thermo Scientific DyNAmo ColorFlash SYBR Green qPCR kit (F-416). The forward and reverse primers used for lair-1 PCR amplification were 5'- TCT CCT CCT CCT GGT CCT CTT C-3' and 5'- GCC TTG TCT GCT GTC CTC TCT A-3'. Housekeeping gene GAPDH was used as the internal control, the primers used for GAPDH were 5' - GTC TCC TCT GAC TTC AAC AGC G -3' and 5'- ACC ACC CTG TTG CTG TAG CCA A -3'. The relative LAIR-1 mRNA expression levels were normalized against GAPDH using the comparative ΔΔCt method and relative fold change of gene was calculated by the equation 2^-ΔΔCt.

Protein level was detected by Western blotting analysis. Cells were lysed using RIPA lysis buffer (Beyotime, China). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membrane, blocked with 5% skim milk. The membrane was probed with a monoclonal mouse anti-LAIR1 antibody [lc12] (1: 500) (ab14826) followed by HRP–conjugated goat anti-mouse antibody (1: 6000) incubation. Protein bands were detected by an ECL kit (Pierce, USA) according to the manufacturer's instructions. The immunoblot of GAPDH was used as a loading control.

Chromatin immunoprecipitation (CHIP)

Jurkat and COC1 cells were used for CHIP experiments. The proteins were cross-linked to the DNA by treating cells with 1% formaldehyde for 10 minutes. The cross-linking reaction was then terminated by the addition of glycine to a final concentration of 0.125 M. The cells were lysed, and cross-linked chromatin was sonicated using a Biorupter (Diagenode) to generate DNA-fragments of approximately 200 to 1000 bp in length. Anti-ETS-2, which was used to precipitate the chromatin fragments were pre-bound to Biomag^TM magnetic beads (Bangs laboratories. Inc). The beads were added to the cell lysates and incubated overnight. The chromatin-antibody complex was then precipitated using a One-Day Chromatin immunoprecipitation kit (Millipore) and digested with proteinase K and RNase to remove the proteins and RNAs. The CHIP DNA was analyzed using PCR amplification. The primers flanking the LAIR-1 gene core promoter were: 5’-ACG GTC ATC TGC CTC TAA GC-3’ (forward) and 5’-CAC CAA TGC AAG GAC AGA ACT C-3’ (reverse). The resultant PCR products were analyzed by 2% agarose gel electrophoresis.

Electrophoretic mobility shift assays (EMSA)

Nuclear extracts were prepared from Hela cells using a nuclear protein preparation kit (Viagene Biotech Co., Ningbo, China). Hela cells were used because of high expression of ETS-2 [23]. EMSA assays were performed using the Complete Non-radioactive EMSA kit (Viagene Biotech Co., Ningbo, China), according to the manufacturer’s instructions and a biotin end-labeled DNA duplex of sequence containing ETS-2 putative binding site A (5’- TCT CAC TAG TGG GAA GGG TGG AAA GAA CAG-3’; 5’-CTG TTC TTT CCA CCC TTC CCA CTA GTG AGA-3’ ), site B (5’-GGA CAG AAA GCT CTT CCT CTT GTG TGA GGC-5’; 5’-GCC TCA CAC AAG AGG AAG AGC TTT CTG TCC-3’) or site C (5’-CGG GCA GCA CAT CCT GTC TGC TTG TGT c-3’; 5’-GAC ACA AGC AGA CAG GAT GTG CTG CCC-3’). Briefly, nuclear protein samples were incubated for 15 minutes at 4°C with 1.0 μg/μL poly (dI-dC) and biotin-labeled and non-labeled competitor oligonucleotide probes in 10× binding buffer. The resulting DNA-protein complex was separated from free oligonucleotide on a 6% polyacrylamide gel, and the samples were then electrotransferred to a positively charged nylon membrane. The membrane was immediately cross-linked for 15 min on a UV transilluminator equipped with 312 nm bulbs. A chemiluminesce method that utilized a luminol/enhancer solution and a stable peroxide solution was used for detection, and the membranes were exposed to x-ray films for 2-5 min before development.